Base Interactions in Low Molecular Weight

Ind. Eng. Chem. Res. 1996, 35, 1097-1106

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Utilization of Soft Acid/Base Interactions in Low Molecular Weight Biochemical Separations Antonio A. Garcıá,*,† Dong-Hoon Kim,‡ and Dale R. Miles§ Department of Chemical, Bio & Materials Engineering, Arizona State University, Tempe, Arizona 85287-6006, Hyundai Petrochemical Company, Ltd., 679 Daejuk-Ri, Daesan-EUP, Seosan-Gun, Chungchongnam-Do, Korea, and Pharmacyclics, Inc., 995 East Arques Avenue, Sunnyvale, California 94086

Categorization of acid/base interactions using hard soft acid base (HSAB) theory suggested that sulfur-containing low molecular weight biological molecules could be specifically targeted for reversible complexation using soft metal ions. A viable method of employing soft metal ions for bioseparations is to immobilize Ag(I) and Pt(II) ions using a soft ligand such as thiourea. This immobilization chemistry allows for the use of Ag(I) columns that are stable in the presence of chloride and phosphate ions in the mobile phase, and it enhances the complexation chemistry of Ag(I) and Pt(II) ions toward solutes which are soft bases. Because chloride ions are soft bases, NaCl can be used for competitive elution. However, in amino acid separations, electrostatic and hydrophobic interactions influence the selectivity and capacity of Ag(I) and Pt(II) columns. A detailed study of the effects of Ag(I) ion loading and pH on the retention time of methionine, histidine, and tryptophan illustrates the need for accounting for Lewis acid/base, electrostatic, and hydrophobic interactions in biological molecule separations. Applying reversible chemical complexation in separation processes has been an interest of C. Judson King for over 17 years. While he stressed that finding an appropriate mass-separating agent (i.e., ligand) in order to obtain a high selectivity for the solute of interest was important, his papers usually discussed a topic often ignored in complexation or so-called “affinity” separations. King frequently noted that of utmost importance to the commercial viability of a complexation separation is that conditions be created, whereby the massseparating agent and the solute of interest are fully recovered in their original chemical state. King stressed this concept throughout his papers, noted the importance of keeping chemical consumption down, and anticipated current engineering efforts toward environmentally benign processing. Many of his papers discussed acid-base theories so as to provide useful strategies or models for the selection of mass-separating agents and process conditions for reversing the complex. Our research on soft acid/base interactions was initiated in part because of the realization that removal of solutes (especially ionized ones) from aqueous solution would have a higher chance for reversibility if the chemical complexation did not deal with interactions in which water plays an active or competitive role. In the theory of hard and soft acids and bases (HSAB) proposed by Pearson (1967, 1968a,b, 1987), water can be considered as both hard acid and hard base. HSAB theory suggests that hard acids prefer to complex with hard bases, while soft acids prefer soft bases. HSAB theory has been applied more recently to metal ions for predicting cation-exchange selectivity (Xu and Harsh, 1990a,b). Soft acid/base interactions occur in aqueous solution without active participation by water molecules in such mechanisms as complex solvation, hydrogen bonding, or proton transfer. Another * To whom comments or questions should be addressed. Phone: (602) 965-8798. Fax: (602) 965-0037. E-mail: ataag@ asuvm.inre.asu.edu. † Arizona State University. ‡ Hyundai Petrochemical Co., Ltd. § Pharmacyclics, Inc.

0888-5885/96/2635-1097$12.00/0

separations benefit of soft acid/base interactions in aqueous solution is that selectivity is higher since water does not compete well for soft acid/base functional groups on the mass-separating agent or solute. After identifying the possible benefits of soft acid/base interactions, the task of creating a viable separation process remained. According to HSAB theory soft metal ions are primarily Ag(I), Cu(I), Au(I), Tl(I), Hg(II), Pt(II), Pd(II), Cd(II), and Pb(II). In HSAB theory, metal ions that are soft acids are typified by having a large atom and a low positive charge and containing several valence electrons that are easily removed or distorted (Pearson, 1967, 1968a,b, 1987). These soft acids prefer to complex with soft bases such as thioether, thiol, and disulfide groups. In contrast, a hard metal ion carries a high positive charge and does not have valence electrons that are easily distorted or removed. Some of the soft metal ions listed above are, of course, quite toxic, while Cu(I) and Ag(I) are difficult to work with since Cu(I) oxidizes readily to the moderately hard acid Cu(II) and Ag(I) is known to precipitate quite readily in chloride and phosphate solutions. Early work on soft metal affinity chromatography by Shaw and West (1980) showed that immobilizing Ag(I) onto a chromatographic support provided a means for selective binding of methionine and ethionine. However, because they used a sulfonic acid resin for immobilizing Ag(I), all proteins and amino acid separations were done using a chloridefree aqueous solution, and the solutes were recovered by elution with acetic acid thus stripping Ag(I) from the column. Our initial use of a strong cation-exchange resin for immobilizing Ag(I) also resulted in a column that was unusable in the presence of phosphate or chloride ions (Garcıá et al., 1994). Employing HSAB theory for selecting a proper immobilization chemistry that could stabilize Ag(I) and provide sufficient complexation strength to bind other metal ions with higher coordination numbers (such as Pt(II)), we chose thiuorea due to its strong interaction with Ag(I) ions (Murray, 1981) and reported some results for Ag(I) and Pt(II) (Garcıá, et al., 1994). Thiourea allowed the use of phosphate buffers and NaCl in the mobile phase without detectable leaching of silver © 1996 American Chemical Society

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Ind. Eng. Chem. Res., Vol. 35, No. 4, 1996

(Garcıá et al., 1994). Pt(II) ions immobilized using thiourea were also found to be stable in the presence of phosphate and imidazole-HCl buffers (Miles and Garcıá, 1995). We refer to this methodology as immobilized soft metal affinity chromatography (ISMAC) in order to illustrate that the use of metal ions in biological separations has expanded to include soft acid/base interactions with biological molecules, which are in this case amino acids. Experimental Equipment and Procedures The resins used in this work are Bio-Gel P-2 poly(acrylamide) gel (Bio-Rad Laboratories, Richmond, CA) (extra fine, 100-200 mesh (wet), 1800 exclusion limit and Model BRX 9013803 strong cation-exchange resin (Bio-Rad Laboratories, Richmond, CA). Immobilization of Ag(I) and Pt(II) on Bio-Gel P-2 was performed using glutaraldehyde (Aldrich Chemical Co., Milwaukee, WI) (25 wt % in water), reagent-grade AgNO3 (Aldrich Chemical Co., Milwaukee, WI), and thiourea (J. T. Baker Chemical, Phillipsburg, NJ) (Garcıá et al., 1994). The BRX 9013803 resin with immobilized Ag(I) was made by contacting the resin with a 1.0 M AgNO3 solution for 24 h at room temperature. All amino acids used were reagent grade (Sigma Chemical Co., St. Louis, Mo). A Pharmacia Augmented FPLC system (Pharmacia, Uppsala, Sweden) was used for all chromatography experiments described here. A silver ion-specific electrode (Orion Research, silver electrode, Model 94-16) was used in order to determine the amount of silver immobilized on the activated gel. A Varian Model 1100 atomic absorbance spectrometer was used for measuring the amount of Pt(II) bound to the Bio-Gel P-2 resin. The slot width was set at 0.2 nm. A wavelength of 265.9 nm using a 10-mA lamp current was used for platinum. A DU-70 spectrophotometer (Beckman Instruments, Fullerton, CA) with a flow-through liquid cell was used for determination of amino acid concentrations exiting the chromatography column. For the BRX 9013803 column with and without Ag(I) ions, glycine was used as the mobile phase, in part because it is the simplest amino acid (i.e., no side chains) but more importantly because phosphate buffers leach and precipitate Ag(I) ions from this resin, causing column plugging. A glass column with dimensions of 0.5 × 2.8 cm (0.55 mL) was used for the experiment. The flow rate through the column was 0.2 mL/min, and the injection volume was 20 µL. A glass column of 0.5 × 5.0 cm packed to a final bed volume of 1.1 mL was used at room temperature for all experiments using Bio-Gel P-2 resin. The mobile phases for the Bio-Gel P-2 column were prepared with (1) a 0.05 M sodium acetate/acetic acid buffer solution for pH 4.0 and 5.0 and (2) a 0.05 M sodium phosphate, di/monobasic buffer solution for pH 7.0. Each mobile phase interferes with the UV spectra of the samples especially

Table 1. Selected Wavelengths for Amino Acids for Different Mobile Phases mobile phases λ, nm amino acids

pH 4.0

pH 4.0 w/NaCla

glutamic acid glutamine arginine methionine histidine phenylalanine tyrosine tryptophan

209 209 210 214 227 220 275 241

210 210 210 214 227 220 275 241

a

pH 5.0

pH 7.0

pH 7.0 w/NaCla

212 212 210 214 227 220 275 241

201 205 209 206 219 221 275 241

204 204 209 207 224 221 274 241

The concentration of NaCl in the mobile phase is 0.25 M.

at short wavelengths. The UV wavelength was selected in order to optimize the signal/noise ratio for each amino acid and mobile phase. Even the same amino acid has a maximum absorbance at different wavelengths when the mobile phase is changed. Glutamic acid, glutamine, and arginine absorb very weakly in the UV range. A wavelength was chosen for these amino acids where the UV absorbance is a maximum value, even though it is in the region of interference by the mobile phase itself. Methionine, histidine, and phenylalanine absorb moderately in the UV range; the wavelength selected for methionine was for its maximum absorbance. The wavelengths for histidine and phenylalanine were chosen where both of them absorb at approximately the same strength as methionine. Tyrosine and tryptophan absorb in the UV range quite strongly. The wavelength for tyrosine was chosen as 274 or 275 nm which is the apex value for the peak in that region. The wavelength for tryptophan detection was chosen as 241 or 242 nm, where the UV absorbance is at a minimum because tryptophan absorbs too strongly. The selected wavelengths for the amino acids studied at different pH values are shown in Table 1. Experimental Results and Discussion Amino Acid Chromatograms Using Ag(I) on BRX 9013803 Cation Exchanger. Figures 1 and 2 show the retention times of various amino acids using the strong acid cation exchanger BRX 9013803 without and with Ag(I). Histidine, methionine, and cysteine are very strongly retained on the Ag(I) form of BRX 9013803 (capacity of 0.12 mmol of Ag(I)/mL of resin) and were not eluted during the course of the experiment. Tryptophan is also strongly retained by the Ag(I) form of the resin, but it is nearly fully eluted during the experiment and would have been fully removed from the column if the experiment were continued longer. On the basis of these data, it would appear that the soft metal ion Ag(I) cannot be readily used to distinguish among amino acids containing soft functional groups and those containing moderately hard groups (i.e., histidine). Moreover, the only buffer that can be used in this separation

Table 2. Dependence of Ag(I) Loadings of Bio-Gel P-2 Resin on the Reaction Time in the Resin Activation Step reaction between glutaraldehyde (GA) and Bio-Gel P-2 resin

a

polymn time of GA (h)a

reaction temp (°C)

24 24 24 24

50 50 50 50

symbol, reaction time (h) GA-06, 6 GA-12, 12 GA-18, 18 GA-24, 24

reaction between thiourea and glutaraldehyde activated resin reaction temp (°C)

reaction time (h)

final loading of Ag(I) on resin (mmol of Ag(I)/mL of resin)

50 50 50 50

24 24 24 24

0.037 0.062 0.097 0.141

Polymerization was performed by keeping a 25 wt % solution of glutaraldehyde at 70 °C.

Ind. Eng. Chem. Res., Vol. 35, No. 4, 1996 1099

Figure 1. Chromatographic response peaks of amino acids on the original form of BRX 9013803. Column volume was 0.55 mL (0.5 × 2.8 cm), and the flow rate was 0.2 mL/min.

Figure 2. Chromatographic response peaks of amino acids on the Ag+ form of BRX 9013803 with 0.12 mmol of Ag(I)/mL of resin. Column volume was 0.55 mL (0.5 × 2.8 cm), and the flow rate was 0.2 mL/min.

is glycine because 38% of the Ag(I) ions are leached from the resin by passing 0.05 M phosphate buffer (pH 7) at a flow rate of 0.2 mL/min for 100 min (Kim, 1994). Amino Acid Chromatograms Using Ag(I) on BioGel P-2. Using thiourea to immobilize Ag(I) onto BioGel P-2 yielded dramatically different results. Although these results were reported elsewhere (Garcıá et al., 1994), they are summarized here for comparison. Phenylalanine, tyrosine, and tryptophan were retained on the original Bio-Gel P-2 column, while all the other amino acids were not retained. Tyrosine and tryp-

tophan were retained for somewhat longer times on the Ag(I) column than on the original form of the resin. This is likely due to the hydroxyl group of tyrosine and the indole group of tryptophan interacting weakly with Ag+ immobilized on the resin. Histidine was retained more strongly on the Ag+ form of the resin compared with the original resin primarily due to the imidazole group in its side-chain interacting with Ag+ on the resin. Methionine was also retained strongly on the Ag(I) form of the resin. However, in sharp contrast to the results with the Ag(I) form of BRX 9013803, methionine and

1100


Figure 3. Chromatographic response peaks of cystine and cysteine on Bio-Gel P-2 resin (original and Ag(I) form). (Both cystine and cysteine retained strongly in the column (0.1mmol of Ag(I)/mL of resin) and were not eluted from the column (during a 2 h run).) Cysteine is abbreviated as Cys. Column: 0.5 × 2.8 cm (0.55 mL), original form Bio-Gel P-2 Resin and 0.10 mmol of Ag+/mL of Bio-Gel P-2 resin. Mobile phase: 0.05 M sodium phosphate buffer, pH 7.0. Flow rate: 0.2 mL/min. Sample loop: 20 µL.

histidine were eluted from the column. A more detailed description of these and other results are provided in the paragraphs below. The chromatograms for cysteine and cystine using the original form of Bio-Gel P-2 and the Ag(I) form of BioGel P-2 are shown in Figure 3. Both cysteine and cystine were not retained on the original form of the resin. However, both cysteine and cystine were retained strongly on the Ag(I) form of the Bio-Gel P-2 resin (0.1 mmol of Ag(I)/mL of resin). Even after a 2 h experiment at a flow rate of 0.2 mL/min (72 column volumes), both cysteine and cystine were not eluted. Cysteine has a thiol group in its side chain and can interact strongly with Ag+ on the resin. These results indicate that the disulfide group of cystine also interacts strongly with the immobilized Ag+ on the thiourea-activated Bio-Gel P-2 resin. This result differs from cystine chromatography on the Ag+ form of the BRX 9013803 resin (Figure 2). Experiments were also performed at lower amounts of Ag(I) loading on the glutaraldehyde-thioureaactivated Bio-Gel P-2 resin to see if cysteine and cystine could be eluted from the column. A different resin containing 0.002 mmol of Ag+/mL resin was used, and chromatograms for histidine, methionine, cystine, and cysteine are shown in Figure 4. Cystine has the same retention time as histidine and methionine. Cysteine was retained more strongly on the Ag+ form of the BioGel P-2 resin than histidine, methionine, or cystine. This result shows that the thiol group of cysteine interacts more strongly with Ag+ on the resin than the thioether group of methionine, the disulfide group of cystine, or the imidazole group of histidine. Other examples which illustrate the change in retention behavior due to Ag(I) are given in Figures 5 and 6. Figure 5 gives the chromatograms of glutamic acid, glutamine, and arginine on the original form of the BioGel P-2 resin at pH 5.0. The retention behaviors for

each of the three amino acids are quite similar in this figure. However, Figure 6 shows the chromatograms of arginine, glutamine, and glutamic acid on the Ag(I) form of the resin (0.097 mmol of Ag+/mL of resin) at pH 5.0. An important factor for the change in retention behavior of these three amino acids is the net charge that they exhibit at pH 5.0. Since the Ag(I) form of BioGel P-2 carries a fixed charge of +1, glutamic acid is retained at pH 5 due to electrostatic attraction while arginine is repelled. The retention time of glutamine was not affected by Ag(I) ions on the resin because glutamine has a small net charge at pH 5.0. These results serve to illustrate that electrostatic interactions play a role in solute retention on an immobilized Ag(I) column and that these interactions must be taken into account in order to properly model retention time behavior for immobilized Ag(I) amino acid chromatography. Effect of NaCl in Ag(I) Chromatography Using Bio-Gel P-2. Figures 7 and 8 show the effect of added salt (NaCl) on the chromatographic response peaks of histidine and methionine at pH 4.0. It should be noted again that, due to immobilization using thiourea, the column performance is not degraded by the presence of chloride ions. In fact, no loss of silver ions was detected under a variety of mobile phase buffer conditions (Garcıá et al., 1994). Retention of histidine was strongly affected at both silver loadings (Kim, 1994) by added salt because the net charge of histidine (ChN ) 1.00) is high and salt decreases electrostatic interactions. Methionine retention at the lower silver loading was affected more than at the higher silver loading at pH 4.0 because chloride ions compete for sites more effectively at lower silver loadings. Better separation can be obtained with a mobile phase which does not contain salt because at pH 4 electrostatic repulsion between Ag+ ions on the resin and histidine can be utilized.


Figure 4. Chromatographic response peaks of amino acids on the Ag(I) form of Bio-Gel P-2. Cysteine is abbreviated as Cys. Column: 0.5 × 5.0 cm (1.0 mL), 0.002 mmol of Ag+/mL of resin. Mobile phase: 0.05 M sodium phosphate buffer, pH 7.0. Flow rate: 0.2 mL/min. Sample loop: 20 µL.

Figure 5. Chromatographic response peaks of glutamic acid, glutamine, and arginine on the original form of the Bio-Gel P-2 resin at pH 5.0. Column: 0.5 × 5.02 cm (0.99 mL). Mobile phase: 0.05 M sodium acetate buffer, pH 5.0. Flow rate: 0.2 mL/min. Sample loop: 20 µL.

Figures 9-11 show the effect of salt on the chromatography of histidine and methionine at pH 7.0. The retention time of methionine at this pH is decreased by addition of 0.25 M NaCl presumably due to both competition with chloride ions for Ag(I) sites and the loss of electrostatic attraction since methionine has a net negative charge at pH 7. Similarly the retention time of histidine also decreased with added salt due to competition with chloride ions for Ag(I) sites. Clearly, chloride ion competition is a very significant effect since

addition of salt should result in shielding of the electrostatic repulsion in the case of histidine, yet at pH 7 the retention time for histidine is drastically reduced with salt addition to the mobile phase. A benefit of salt addition to the mobile phase is peak sharpening, especially at higher loadings of Ag(I) (Kim, 1994). Effect of pH and Ag(I) Loading on Amino Acid Chromatography. Since thiourea immobilization allows for a wider choice of pH buffers and since the Ag(I) loading has a dramatic effect on the retention of some

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Figure 6. Chromatographic response peaks of glutamic acid, glutamine, and arginine on the Ag(I) form of Bio-Gel P-2 (0.097 mmol of Ag+/mL of resin) at pH 5.0. Column: 0.5 × 5.11 cm (1.00 mL). Mobile phase: 0.05 M sodium acetate buffer, pH 5.0. Flow rate: 0.2 mL/min. Sample loop: 20 µL. Table 3. Parameters for Equation 1 Solved by Numerical Fitting (Kim, 1994) amino acid

β′1

β′2

Xc

Xd

SSQb

histidine methioninea tryptophana

16.969 4.8846 7.9665

2286.9 420.75 78.055

1.7265 1.7265 1.7265

1.3051 0.01733 0.01244

3.2162 11.445 1.2679

a Parameters in eq 1 obtained by numerical fitting after fixing Xc to the value for histidine. b SSQ is the sum of the squares for the fit.

amino acids, we investigated the effect of pH and Ag(I) loading on the retention of methionine, tryptophan, and histidine. A mathematical model that rationalizes the experimental data has been reported elsewhere (Kim and Garcıá, 1995), but it is important to briefly summarize the results for this discussion. Equation 1 shows

ht R )

[

(

Xd2 {Xc - (+1)ChN} L 1- 1+ φ[Re] + β′1 × v Xc (X + Ch )2

(

)

d

N

)]

Xd2 {Xc - (+2)ChN} 2 ] + β′ [Ag+ [Ag+ tot 2 tot] Xc (X + Ch )2 d

N

(1)

the resultant four-parameter model for negligible internal or external mass-transfer resistance which was used to describe (1) hydrophobic interactions, (2) specific acid/base interactions; and (3) electrostatic interactions for Ag(I) chromatography of methionine, histidine, and tryptophan. The term ChN is the net charge on the amino acid. This model takes into account only 1:1 or 2:1 Ag(I)-amino acid complexes (Kim and Garcıá, 1995). The parameters in Table 3 were obtained by a fit of the experimental data, provided in Tables 4-6, with eq 1. These parameters enabled the model to follow the experimental data within a reasonably good margin (Kim and Garcıá, 1995). Especially important is the match of the trends at low pH; namely, that the retention times of histidine decrease with decreasing

Table 4. Summary of Retention Data of Tryptophan on the Ag+ Form of Bio-Gel P-2 (Refer to Table 2 for Concentrations of Ag+)

tryptophan pH 4.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 4.0 w/salt GA-12 resin GA-24 resin pH 5.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 w/ salt GA-12 resin GA-24 resin

retention time (min)

column length (L, cm)

normalized retention time (min)

σhtRNa (min)

7.36 7.36 7.66 7.86 8.16

5.02 5.05 5.08 5.11 5.04

7.33 7.29 7.54 7.69 8.10

0.080 0.080 0.082 0.084 0.089

7.46 8.06

5.03 5.11

7.42 7.89

0.081 0.086

7.36 7.46 7.76 8.16 8.56

5.02 5.05 5.08 5.11 5.04

7.33 7.39 7.64 7.98 8.49

0.080 0.081 0.084 0.087 0.093

7.36 7.66 8.56 11.06 12.46

5.20 4.99 5.06 5.10 5.11

7.08 7.68 8.46 10.84 12.19

0.077 0.084 0.093 0.119 0.133

7.56 8.01

5.11 5.12

7.40 7.82

0.081 0.086

a Note: σ htRN is the standard deviation of the retention time (Kim, 1994).

pH. A switch in the retention order between methionine and histidine occurs at pH 5. Methionine is retained longer than histidine at pH 5, while at pH 7 the reverse is true (Kim and Garcıá, 1995). Immobilized Soft Metal Affinity Chromatography (ISMAC) Compared with Chromatography Using Moderately Hard Metal Ions. Table 7 summarizes retention time data for the chromatography of several amino acids using silver and platinum ions along


Figure 7. Chromatographic response peaks of histidine and methionine on the Ag+ form of Bio-Gel P-2 (refer to Table 2 for concentrations of Ag+) at pH 4.0. Column: 0.5 × ∼5.0 cm (∼1.0 mL). Mobile phase: 0.05 M sodium acetate buffer, pH 4.0. Flow rate: 0.2 mL/min. Sample concentration: 0.005 M. Sample loop: 20 µL. Table 5. Summary of Retention Data of Methionine on the Ag+ Form of Bio-Gel P-2 (Refer to Table 2 for Concentrations of Ag+)

methionine pH 4.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 4.0 w/salt GA-12 resin GA-24 resin pH 5.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 w/salt GA-12 resin GA-24 resin




σhtRNa (min)

4.16 5.06 6.26 6.66 7.76

5.02 5.05 5.08 5.11 5.04

4.14 5.01 6.16 6.52 7.70

0.045 0.055 0.067 0.071 0.084

5.76 7.96

5.03 5.11

5.73 7.79

0.063 0.085

4.26 5.26 6.51 7.16 8.81

5.02 5.05 5.08 5.11 5.04

4.24 5.21 6.41 7.01 8.74

0.046 0.057 0.070 0.077 0.096

4.46 5.81 9.26 19.46 32.56

5.20 4.99 5.06 5.10 5.11

4.29 5.82 9.15 19.08 31.86

0.047 0.064 0.100 0.209 0.348

5.81 7.66

5.11 5.12

5.68 7.48

0.062 0.082

Table 6. Summary of Retention Data of Histidine on the Ag+ Form of Bio-Gel P-2 (Refer to Table 2 for Concentrations of Ag+)

histidine pH 4.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 4.0 w/salt GA-12 resin GA-24 resin pH 5.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 original form GA-06 resin GA-12 resin GA-18 resin GA-24 resin pH 7.0 w/salt GA-12 resin GA-24 resin




σhtRNa (min)

3.76 3.46 3.16 2.96 2.91

5.02 5.05 5.08 5.11 5.04

3.75 3.43 3.11 2.90 2.89

0.041 0.037 0.034 0.032 0.032

4.61 4.76

5.03 5.11

4.58 4.66

0.050 0.051

3.96 4.16 4.11 3.86 3.76

5.02 5.05 5.08 5.11 5.04

3.94 4.12 4.05 3.78 3.73

0.043 0.045 0.044 0.041 0.041

4.46 5.86 9.26 19.16 32.16

5.20 4.99 5.06 5.10 5.11

4.29 5.87 9.15 18.78 31.47

0.047 0.064 0.100 0.205 0.344

5.31 5.96

5.11 5.12

5.20 5.82

0.057 0.064



with a comparison of results using other metal ions reported by other researchers. A more detailed discussion of Pt(II) chromatography is provided elsewhere (Garcıá et al., 1994). Upon review of ISMAC and metal affinity data for transition-metal ions, several conclusions can be listed: (1) for both Ag(I) and Pt(II) at pH 7.0, methionine is strongly retained; (2) for Ag(I) (at a pH ) 4.7) and for Pt(II), histidine is not the most

strongly retained amino acid as is the case with Cu(II) and Zn(II) for pH ) 4-8; and (3) tryptophan is only slightly retained using Ag(I), while for Pt(II) it shows no retention time difference over the original or modified naked gel. These results illustrate key differences in complexation chemistry between transition-metal ions which are moderately hard Lewis acids and silver and platinum ions which are soft acids.

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Figure 8. Chromatographic response peaks of histidine and methionine on the Ag+ form of Bio-Gel P-2 (refer to Table 2 for concentrations of Ag+) at pH 4.0 with added NaCl. Column: 0.5 × ∼5.0 cm (∼1.0 mL). Mobile phase: 0.05 M sodium acetate buffer w/0.25 M NaCl, pH 4.0. Flow rate: 0.2 mL/min. Sample concentration: 0.005 M. Sample loop: 20 µL.

Figure 9. Chromatographic response peaks of methionine on the Ag+ form of Bio-Gel P-2 (refer to Table 3 for concentrations of Ag+) at pH 7.0. Column: 0.5 × ∼5.0 cm (∼1.0 mL). Mobile phase: 0.05 M sodium phosphate buffer, pH 7.0. Flow rate: 0.2 mL/min. Sample concentration: 0.005 M. Sample loop: 20 µL.

Summary and Conclusions Soft acid/base interactions can be used to selectively and reversibly complex with amino acids. However, amino acid complexation with immobilized Ag(I) and Pt(II) ions is not solely due to acid/base interactions. The charge on the amino acid, which depends on the pH, and hydrophobic interactions must be considered when designing separation processes using soft metal ions.

Immobilization chemistry can affect (1) capacity; (2) selectivity; and (3) metal ion stability. The use of a soft base ligand such as thiourea allows the use of buffers that are commonly encountered in commercial bioseparation processes. Lowering the Ag(I) ion loading allows strongly interacting solutes to be eluted from the column and provides a method for separation among sulfurcontaining amino acids.


Figure 10. Chromatographic response peaks of histidine on the Ag+ form of Bio-Gel P-2 (refer to Table 2 for concentrations of Ag+) at pH 7.0. Column: 0.5 × ∼5.0 cm (∼1.0 mL). Mobile phase: 0.05 M sodium phosphate buffer, pH 7.0. Flow rate: 0.2 mL/min. Sample concentration: 0.005 M. Sample loop: 20 µL. Table 7. Comparison of IMAC Retention Factors (Ve/Vt) of Selected Amino Acidsa amino acid asparagine glutamic acid proline phenylalanine tyrosine tryptophan histidine methionine a

Cu2+ (pH ) 7)

Ni2+ (pH ) 7)

2.31 1.17

4.9 0.8

1.95 5.71 >40 2.28

6.0 9.3 >20 4.7

Zn2+ (pH ) 7)

1.24 2.54

Fe2+ (pH ) 7)

1.29 4.15

Ag+ (pH ) 7) 1.0 0.9 1.0 1.2 1.6 3.7 13 8

Ag+ (pH ) 4.7)

Pt2+ (pH ) 7)

1.1 1.4

1.1 1.3 1.7 1.2 >5

Ni2+ data are from Hemden and Porath (1985), while the Cu2+, Zn2+, and Fe2+ data are from Rassi and Horvath (1986).

Immobilized soft metal affinity chromatography (ISMAC) broadens the use of metal ions in bioseparations. Other potential uses of ISMAC for low molecular weight biochemical separations include recovery of sulfurcontaining metabolites such as biotin, cephalosporins, and β-lactam antibiotics. It is also expected that ISMAC will find utility in the purification and recovery of sulfurcontaining peptides. Acknowledgment This work was supported by grants from the National Science Foundation (BCS-9009301) and Genencor International (South San Francisco, CA). Nomenclature + [Ag+ tot] ) total concentration of Ag within the resin ChN ) net charge of amino acids at certain pH GA ) glutaraldehyde GA-06 ) Ag+-form poly(acrylamide) resin prepared with 6 h of reaction in the GA activation step (refer to Table 2) GA-12 ) Ag+-form poly(acrylamide) resin prepared with 12 h of reaction in the GA activation step (refer to Table 2)

GA-18 ) Ag+-form poly(acrylamide) resin prepared with 18 h of reaction in the GA activation step (refer to Table 2) GA-24 ) Ag+-form poly(acrylamide) resin prepared with 24 h of reaction in the GA activation step (refer to Table 2) HSAB ) hard and soft acids and bases IMAC ) immobilized metal ion affinity chromatography ISMAC ) immobilized soft metal ion affinity chromatography L ) bed length of the column [Re] ) concentration of hydrophobically active sites within resin htR ) retention time of chromatographic peak v ) interstitial velocity of fluid Xc ) parameter that interprets the amount of contribution from the magnitudes of electrostatic attraction or repulsion according to the net charge of the solute Xd ) parameter which takes account of the effect of distance between the sample and Ag+ ion on the resin Greek Symbols β1 ) equilibrium constant β′1 ) parameter defining the contribution to the (1:1) equilibrium constant for specific interactions between Ag(I) and an amino acid

1106


Figure 11. Chromatographic response peaks of histidine and methionine on the Ag+ form of Bio-Gel P-2 (refer to Table 2 for concentrations of Ag+) at pH 7.0 with added NaCl. Column: 0.5 × ∼5.0 cm (∼1.0 mL). Mobile phase: 0.05 M sodium phosphate buffer w/0.25 M NaCl, pH 7.0. Flow rate: 0.2 mL/min. Sample concentration: 0.005 M. Sample loop: 20 µL.

β′′1 ) parameter defining the contribution to the (1:1) equilibrium constant for nonspecific charge interactions between Ag(I) and an amino acid β2 ) equilibrium constant β′2 ) parameter defining the contribution to the (2:1) equilibrium constant for specific interactions between Ag(I) and an amino acid β′′2 ) parameter defining the contribution to the (2:1) equilibrium constant for nonspecific charge interactions between Ag(I) and an amino acid ) voidage of adsorbent bed φ ) equilibrium constant σhtRN ) standard deviation of the retention time, min

Literature Cited Garcia, A. A.; Kim, D. H.; Miles, D. Immobilization of silver and platinum ions for metal affinity chromatography. React. Polym. 1994, 23, 249. Hemdan, E. S.; Porath, J. Development of immobilized metal affinity chromatography: II. interaction of amino acids with immobilized nickel iminodiacetate. J. Chromatogr. 1985, 323, 255. Kim, D. H. Separation of amino acids by immobilized soft metal affinity chromatography using Ag(I). Ph.D. Dissertation, Arizona State University, Tempe, AZ, 1994. Kim, D. H.; Garcia, A. A. Retention behavior of amino acids using immobilized Ag(I) chromatography. Biotechnol. Prog. 1995, 11, 465. Miles, D. R. Amino acid and protein chromatography using immobilized platinum (II) ions. Ph.D. Dissertation, Arizona State University, Tempe, AZ, 1994. Miles, D.; Garcia, A. A. Separation of biotin labeled proteins from their unlabeled counterparts using immobilized platinum affinity chromatography. J. Chromatogr. 1995, 702, 173.

Murray, S. G.; Hartley, F. R. Coordination chemistry of thioethers, selenoethers, and telluroethers in transition-metal complexes. Chem. Rev. 1981, 81, 365. Pearson, R. G. Hard and soft acids and bases. Chem. Br. 1967, 3, 103. Pearson, R. G. Hard and soft acids and bases, HSAB, part I. J. Chem. Educ. 1968a, 45 (10), 581. Pearson, R. G. Hard and soft acids and bases, HSAB, part II. J. Chem. Educ. 1968b, 45 (10), 643. Pearson, R. G. Recent advances in the concept of hard and soft acids and bases. J. Chem. Educ. 1987, 64, 561. Rassi, Z. E.; Horvath, C. Metal chelate interaction chromatography of proteins with iminodiacetic acid bonded stationary phases on silica support. J. Chromatogr. 1986, 359, 241. Shaw, D. C.; West C. E. The isolation of methionine and ethionine by silver ligand chromatography and application to methioninecontaining peptides. J. Chromatogr. 1980, 200, 185. Xu, S.; Harsh, J. B. Monovalent cation selectivity quantitatively modeled according to hard/soft/acid/base theory. Soil Sci. Soc. Am. J. 1990a, 54, 357. Xu, S.; Harsh, J. B. Hard and soft acid-base model verified for monovalent cation selectivity. Soil Sci. Soc. Am. J. 1990b, 54, 1597.

Received for review June 2, 1995 Revised manuscript received February 6, 1996 Accepted February 8, 1996X IE950331Y

X Abstract published in Advance ACS Abstracts, March 15, 1996.

Base Interactions in Low Molecular Weight

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